Angiogenesis
Angiogenesis is a complex process in which capillary blood vessels grow in an ordered sequence of events (Folkman and Klagsbrun, Science 235, 442-447, 1987; Folkman and Shing, J. Biol. Chem. 267, 10931-10934, 1992). A substantial body of evidence supports the hypothesis that tumor angiogenesis is fundamental for the growth and metastasis of solid tumors (Folkman and Klagsbrun ibid., 1987; Weidner et al. Amer. J. Pathol. 143, 401-409, 1993; O'Reilly et al. Cell 79, 316-328, 1994). Indeed, the majority of clinical tumors are not even clinically detectable until after the occurrence of neovascularization, whose induction in solid tumors is mediated by one or more angiogenic factors.
Furthermore, angiogenesis is also important in a number of other pathological processes, including, but not limited to, arthritis, psoriasis, diabetic retinopathy, retinopathy of prematurity, macular degeneration, scleroderma, hemangioma, retrolental fibroplasia, abnormal capillary proliferation in hemophiliac joints, prolonged menstruation and other disorders of the female reproductive system. Thus, methods of blocking angiogenesis are clearly necessary.
The basic mechanism of angiogenesis may be outlined briefly as follows. When a new capillary sprouts from the side of a venule, endothelial cells degrade the basement membrane, migrate toward an angiogenic source, proliferate, form a lumen, join the tips of two sprouts to generate a capillary loop, and manufacture a new basement membrane (Folkman, Perspectives in Biology and Medicine, 29, 1-36, 1985).
Degradation and remodeling of the extracellular matrix (ECM) are essential processes for the mechanism of angiogenesis. In addition, ECM components synthesized by endothelial cells (i.e., collagens, laminin, thrombospondin, fibronectin and SPARC) function to regulate endothelial cell growth, migration and shape (Bischoff, Trends Cell Biol. 5, 69-744, 1995). Bovine aortic endothelial cells (BAE), while undergoing sprouting and tube formation, synthesize collagen and SPARC. It has been proposed that type I collagen may be involved in directing the migration and assembly of BAE cells (Iruela-Arispe et al. Lab. Invest. 64, 174-186, 1991).
In order to treat angiogenesis related disorders, several inhibitors of the angiogenesis mechanism are being studied, including platelet factor 4, the fumagillin derivative AGH 1470, Interferon (.alpha..sub.2 a, thrombospondin, angiostatic steroids, and angiostatin (Folkman ibid., 1995; O'Reilly et al., ibid., 1994). In addition, anti-estrogens have also been shown to inhibit angiogenesis (Garliardi and Collins, Cancer Res. 53, 533-535, 1993). Unfortunately, many of these inhibitors all share the property of being relatively non-specific in their effects and, therefore, potentially toxic. A more specific inhibitor would be most useful, particularly an inhibitor that would selectively block an underlying mechanism of angiogenesis without adversely affecting other physiological functions. Furthermore, many of the compounds that are now being evaluated as anti-angiogenic agents are proteins (e.g., antibodies, thrombospondin, angiostatin and platelet factor IV) which generally suffer from poor bioavailability and are readily degraded in the body. Hence, they must be administered in high doses and frequencies.
There is, thus, a widely recognized unmet need for an inhibitor of angiogenesis which specifically blocks the proliferation of vascular structures without substantially affecting other physiological processes--including an inhibitor of angiogenesis associated with tumor growth or progression.
Permanently Charged Steroid Hormones and Their Antagonists
Pharmaceutical therapies for breast cancer currently consists of hormonal and cytotoxic agents. Hormonal therapy was developed because, in many women, breast cancer cells have receptors for the steroid hormone estrogen. The growth of these estrogen receptor-positive cancer cells can be stimulated by estrogen. Anti-estrogen therapy attempts to reduce or stop the synthesis of estrogen or to block the action of estrogen on the cancer cell.
Among all hormonals, tamoxifen (U.S. Pat. No. 4,536,516) holds a prevalent position. originally used as an anti-estrogen to treat breast cancer in patients with estrogen receptor-positive tumors, the drug was also found to slow the growth of breast cancer in women with estrogen receptor-negative tumors. Tamoxifen is, therefore, useful in most patients. The anti-estrogen tamoxifen is particularly effective in delaying recurrence in breast cancer patients and in the palliative treatment of advanced metastatic breast cancer. It is also useful in the treatment of gliomas and hepatomas as well as endometrial, uterine, ovarian and prostatic neoplasms (Litherland, S. et al. Cancer Treatment Reviews, 15, 183, 1988; Jordan, C., Br. J. Pharmacol., 110, 507, 1993).
Anti-estrogens, including tamoxifen, compete with estrogen for receptor sites in cancerous tissues. Occupancy of the receptor site by an anti-estrogen fails to elicit the full spectrum of transcriptional actions generated by estrogens and, thus, blocks their activity. It is generally believed that estrogens function by first binding to the target cell cytosolic receptors, and then moving into the cell nucleus, where they affect DNA transcription.
Considerable effort has been invested in the development of novel tamoxifen analogs presumed to have improved therapeutic potential, by virtue of their increased selectivity as anti-estrogenic compounds (e.g., U.S. Pat. No. 4,973,755; EP 0 168,175) or their higher affinity for the estrogen receptor (WO 92/06068).
Hydrophilic compounds and particularly compounds with ionic charges (cationic or anionic) are often very poorly distributed into the CNS and brain since a lipophilic barrier (the blood-brain barrier) exists. One method for creating a permanent charge on a drug is the incorporation of a quaternary ammonium salt (nitrogen with four hydrocarbon groups attached). Tamoxifen and other anti-estrogens that contain an amino group can be quaternized (converted to a quaternary ammonium group). Such quaternization results in imparting a permanent positive charge to the parent molecule which should effectively reduce the molecule's penetration across physiological membranes which are inherently lipophilic and resistant to penetration of ions, particularly large ions.
Several quaternary salts of tamoxifen have been prepared and described in scientific publications (Jarman et al., Anticancer Drug Design, 1, 259, 1986). When tested in vitro, these derivatives were reported not to halt the proliferation of breast tumor cell lines grown in culture. These compounds were, therefore, predicted to be of no therapeutic value in vivo.
WO 95/26720 disclosed that unexpectedly ionic derivatives of the anti-estrogen tamoxifen, which were predicted to be of no value in vivo on the basis of their lack of activity in vitro, are, in fact, more active as anticancer agents in vivo than the parent compound. This invention is applicable, in principle, to a wide variety of other anti-estrogens where adverse side-effects may be reduced or eliminated by preventing access of the drugs to the CNS.
In a study of MCF-7 human breast cancer implanted in nude mice, TMI proved to be significantly more potent than tamoxifen in its anticancer action. TMI induced tumor regression that began almost immediately upon dose initiation and which resulted in complete regression of the implanted cancer in 40% of animals tested. The parent compound, tamoxifen, merely slowed tumor growth in that study (Cancer Res. 56, 4238, 1996).
While tamoxifen and other anti-estrogens have been reported to have angiostatic activity in tumors, the mechanism of inhibition of angiogenesis is not clear (Cancer Res. 54, 5511, 1994; Cancer Res. 53, 533, 1993).